Canola is a genetic variation of rapeseed developed by Canadian plant breeders specifically for its oil and meal attributes, particularly its low level of saturated fat. “Canola” generally refers to plants of Brassica species that have less than 2% erucic acid (Δ13-22:1) by weight in seed oil and less than 30 micromoles of glucosinolates per gram of oil free meal. Typically, canola oil may include saturated fatty acids known as palmitic acid and stearic acid, a monounsaturated fatty acid known as oleic acid, and polyunsaturated fatty acids known as linoleic acid and linolenic acid. These fatty acids are sometimes referenced by the length of their carbon chain and the number of double bonds in the chain. For example, oleic acid is sometimes referred to as C18:1 because it has an 18-carbon chain and one double bond, linoleic acid is sometimes referred to as C18:2 because it has an 18-carbon chain and two double bonds, and linolenic acid is sometimes referred to as C18:3 because it has an 18-carbon chain and three double bonds. Canola oil may contain less than about 7% total saturated fatty acids (mostly palmitic acid and stearic acid) and greater than 60% oleic acid (as percentages of total fatty acids). Traditionally, canola crops include varieties of Brassica napus and Brassica rapa. Recently, a canola quality Brassica juncea variety, which has oil and meal qualities similar to other canola types, has been added to the canola crop family (U.S. Pat. No. 6,303,849, to Potts et al., issued on Oct. 16, 2001; U.S. Pat. No. 7,423,198, to Yao et al.; Potts and Males, 1999; all of which are incorporated herein by reference).
The fatty acid composition of a vegetable oil affects the oil's quality, stability, and health attributes. For example, oleic acid (a C18:1 monounsaturated fatty acid) has been recognized to have certain health benefits, including effectiveness in lowering plasma cholesterol levels, making higher levels of oleic acid content in seed oil (>70%) a desirable trait. Further, not all fatty acids in vegetable oils are equally vulnerable to high temperature and oxidation. Rather, the susceptibility of individual fatty acids to oxidation is dependent on their degree of unsaturation. For example, linolenic acid (C18:3), which has three carbon-carbon double bonds, oxidizes 98 times faster than oleic acid, which has only one carbon-carbon double bond, and linoleic acid, which has two carbon-carbon double bonds, oxidizes 41 times faster than oleic acid (R. T. Holman and O. C. Elmer, “The rates of oxidation of unsaturated fatty acid esters,” J. Am. Oil Chem. Soc. 24, 127-129 1947. For further information regarding the relative oxidation rates of oleic, linoleic and linolenic fatty acids, see Hawrysh, “Stability of Canola Oil,” Chap. 7, pp. 99-122, CANOLA AND RAPESEED: PRODUCTION, CHEMISTRY, NUTRITION, AND PROCESSING TECHNOLOGY, Shahidi, ed., Van Nostrand Reinhold, N.Y., 1990, incorporated by reference herein.
The “stability” of a vegetable oil can be defined as the resistance of the oil to oxidation and to the resulting deterioration due to the generation of products causing rancidity and decreasing food quality. Under identical processing, formulation, packaging and storage conditions, the major difference in stability between different vegetable oils is due to their different fatty acid profiles. High oleic acid content vegetable oil is therefore preferred in cooking applications because of its increased resistance to oxidation in the presence of heat. Poor oxidative stability brings about, for example, shortened operation times in the case where the oil is used as a fry oil because oxidation produces off-flavors and odors that can greatly reduce the marketable value of the oil. For these reasons, high oleic acid and low linolenic acid may be desirable traits in plant oils.
Plants synthesize fatty acids in their plastids as palmitoyl-ACP (16:0-ACP) and stearoyl-ACP. The conversion of stearoyl-ACP to oleoyl-ACP (18:1-ACP) is catalyzed by a soluble enzyme, the stearoyl-ACP Δ9 desaturase (Shanklin and Somerville, 1991). These acyl-ACPs are either used for glycolipid synthesis in chloroplasts or transported out of chloroplasts into the cytoplasm as acyl-CoAs. Further desaturation of oleic acid occurs only after it is used in the synthesis of glycerolipids and incorporated into membranes, which leads to the synthesis of polyunsaturated fatty acids.
It is widely known by those of skill in the art that the unsaturation of fatty acids in oilseed crops is controlled in part by fatty acid desaturase (FAD) enzymes. FAD enzymes regulate the unsaturation of fatty acids, such as stearic acid (C18:0), oleic acid (C18:1) and linoleic acid (C18:2), through the removal of hydrogen atoms from defined carbons of a fatty acyl chain, creating carbon-carbon double bonds. The synthesis of polyunsaturated fatty acids linoleate (Δ9, 12-18:2) and α-linolenate (Δ9, 12, 15-18:3) begins with the conversion of oleic acid (Δ9-18:1) to linoleic acid, the enzymatic step catalyzed by the microsomal ω-6 oleic acid desaturase (FAD2). The linoleic acid is then converted to co-linolenic acid through further desaturation by ω-3 linoleic acid desaturase (FAD3). There are reports that manipulation of the FAD2 gene through genetic engineering could alter fatty acid profiles. For example, heterologous expression of a soybean fad2 gene in an Arabidopsis mutant line led to dramatic increase in the accumulation of polyunsaturated fatty acids (Heppard et al., 1996). In contrast, in an Arabidopsis mutant line fad2-5, where the transcription of the fad2 gene was decreased significantly due to T-DNA insertion, showed a dramatic increase in the accumulation of oleic acid and a significant decrease in the levels of linoleic acid and linolenic acid (Okuley et al., 1994). These findings suggest that the FAD2 gene plays an important role in controlling conversion of oleic acid to linoleic acid in seed storage lipids.
Significant efforts have been made to manipulate the fatty acid profile of plants, particularly oil-seed varieties such as Brassica spp. that are used for the large-scale production of commercial fats and oils (see, for example, U.S. Pat. Nos. 5,625,130 issued 29 Apr. 1997, 5,668,299 issued 16 Sep. 1997, 5,767,338 issued 16 Jun. 1998, 5,840,946 issued 24 Nov. 1998, 5,850,026 issued 15 Dec. 1998, 5,861,187 issued 19 Jan. 1999, 6,063,947 issued 16 May 2000, 6,084,157 issued 4 Jul. 2000, 6,169,190 issued 2 Jan. 2001, 6,323,392 issued 27 Nov. 2001, and international patent applications WO 97/43907 published 27 Nov. 1997 and WO 00/51415 published 8 Sep. 2000).
Brassica juncea (AA BB genome; n=18) (also referred to herein as “B. juncea”) is an amphidiploid plant of the Brassica genus that is generally thought to have resulted from the hybridization of Brassica rapa (AA genome; n=10) and Brassica nigra (BB genome; n=8). Brassica napus (AA CC genome; n=19) (also referred to herein as “B. napus”) is also an amphidiploid plant of the Brassica genus but is thought to have resulted from hybridization of Brassica rapa and Brassica oleracea (CC genome; n=9). Under some growing conditions, B. juncea may have certain superior traits to B. napus. These superior traits may include higher yield, better drought and heat tolerance and better disease resistance. Intensive breeding efforts have produced plants of Brassica species whose seed oil contains less than 2% erucic acid and whose de-fatted meal contains less than 30 micromoles glucosinolates per gram. The term “canola” has been used to describe varieties of Brassica spp. containing low erucic acid (Δ13-22:1) and low glucosinolates. Typically, canola oil may contain less than about 7% total saturated fatty acids and greater than 60% oleic acid (as percentages of total fatty acids). For example, in the United States, under 21 CFR 184.1555, low erucic acid rapeseed oil derived from Brassica napus or Brassica rapa is recognized as canola oil where it has an erucic acid content of no more than 2% of the component fatty acids, an oleic acid (C18:1) content of over 50.0% by weight, a linoleic acid (C18:2) content of less than 40.0% by weight, and a linolenic acid (C18:3) content of less than 14.0% by weight. In Canada, the addition of Brassica juncea to the canola definition by the Canola Council of Canada set the additional requirements that Brassica juncea canola varieties must produce seeds having an oil comprising an oleic acid content equal to or greater than 55% of total fatty acids in the seeds, and meal derived from Brassica juncea canola seeds must contain less than 1 micromole of allyl (2-propenyl) glucosinolates per gram of oil free meal.
Differences between the oil compositions of Brassica juncea and Brassica napus are well known in the art. For example, Brassica juncea is known to contain differences in various constituents, including, but not limited to, phenolics (e.g., tocopherols), sterols, sulfides, fatty acid constituents, minerals, and isothiocyanates. Brassica juncea also contains volatiles having strong antimicrobial (bacteria and fungi) properties.
Plant breeders have also selected canola varieties that are low in glucosinolates, such as 3-butenyl, 4-pentenyl, 2-hydroxy-3-butenyl or 2-hydroxy-4-pentenyl glucosinolate. Canola quality meal may for example be defined as having a glucosinolate content of less than 30 micromoles of aliphatic glucosinolates per gram of oil-free meal. Currently, the principal commercial canola crops comprise Brassica napus and Brassica rapa (campestris) varieties. U.S. Pat. No. 6,303,849 issued to Potts et al., on 16 Oct. 2001 (incorporated herein by reference) discloses Brassica juncea lines having edible oil that has properties similar to canola. The Brassica juncea lines disclosed therein have a lineage that includes Brassica juncea lines J90-3450 and J90-4316, deposited as ATCC Accession Nos. 203389 and 203390 respectively (both of which were deposited by Agriculture and Agri-Food Canada under the terms of the Budapest Treaty on 23 Oct. 1998 at the American Type Culture Collection, 10801 University Blvd., Manassas, Va. USA 20110-2209).
The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings.